Cross priming amplification of target nucleic acids

ABSTRACT

The present invention relates to methods of amplification of nucleic acid sequences; more particularly, it relates to methods of amplifying target sequences by utilizing cross priming isothermal amplification. The present invention relates to methods of marking the amplification target sequence during the amplification reaction and rapid detection of the target sequence. The present invention also relates to reagent kits for rapid nucleic acid diagnosis and the nucleic acid detection of pathogenic microorganisms such as bacteria, viruses, as well as to diagnoses related to human genetic diseases.

This application claims benefit under 35 USC §119(e) of U.S. Provisionalpatent Application Ser. No. 61/398,798 filed Jul. 1, 2010.

FIELD OF THE INVENTION

The present invention relates to methods of cross primer amplificationof target sequences and the amplification target sequence reagent kitsand the applications.

BACKGROUND OF THE INVENTION

In the past 30 years, pathogenic microorganisms which are difficult tocultivate or can not be cultured have become the main source ofcontagious or infectious diseases. Prior art detection methods usingmarkers such as number of proliferating bacterial colonies, colonypurification separation, external morphology and physiological andbiochemical identification as well as serological identification do notmeet the fast, easy, high-specificity identification requirements oftoday because of their time-consuming, tedious steps and othershortcomings. Therefore, it is increasingly important to correctlyidentify these pathogenic microorganisms which are difficult tocultivate or cannot be cultured and to study these pathogenic bacterianucleic acid structures and molecular characteristics at the molecularbiology level thereby greatly enhancing the detection of such pathogenicbacteria. At present, highly sensitive, highly specific and rapidnucleic acid amplification technology can directly detect clinicalspecimens. However these techniques have been applied more widely in theinfectious disease diagnoses and there is a trend to gradually replacetraditional bacteria or virus cultivation.

Polymerase chain reaction (PCR) technology is the most widely usednucleic acid amplification technology currently employed. At present,the external nucleic acid amplification technology may be divided intotwo types. The first type is characterized by cycling temperatures inthermal insulation spots and includes PCR, ligase chain reaction (LCR),and transcription based amplification systems (TAS). The second typeincludes isothermal amplification systems such as the stranddisplacement amplification (SDA), nucleic acid sequence basedamplification (NASBA), transcription mediated amplification (TMA),rolling circle amplification (RCA), loop-mediated isothermalamplification (LAMP), helicase dependent amplification (HDA). Thesemethods all share the common characteristic that the amplificationreactions are carried out under a uniform temperature so therebysimplifying the instrumentation required for the amplification reaction.

Most of various nucleic acid amplification technologies are coupled tovarious detection methodologies such as electrophoresis, fluorescence,mass spectrometry or direct sequencing so as to detect target sequencesthat are amplified. The detection technologies often involve complicatedoperations, are costly and usually require large-scale equipment thatmust be operated by skilled professionals. Furthermore, these techniquesare often not suitable for broad application and use in many rural thirdworld hospitals. The present invention combines isothermal nucleic acidamplification technology and nucleic acid testing strip rapid detectionfor simple, rapid, low cost detection of pathogens as well as othernucleic acid containing organisms.

As previously described there are several methods of nucleic acidamplification. PCR is accomplished by providing oligonucleotide primersat both sides of the target sequence so as to enzymatically synthesizeseveral target sequence DNA fragments. Each cycle of PCR includes theDNA double strand separation, primer renaturation and an extensionreaction catalyzed by the DNA polymerase which makes newly synthesizedDNA fragments that may again become the templates for next cycle ofamplification thereby giving rise to the exponential amplification ofthe target sequence DNA. At present, PCR technology has been appliedwidely in various aspects in the biotechnology such as detection ofgenetic diseases, cancer diagnoses and prognosis, identification ofbacteria, viruses and fungal infection; and paternity.

LCR is amplification based on the connection capability of the Taqligase, which is able to detect point mutations in a target genesequence. LCR may identify the specific point mutation more readily thanPCR. If there are any point mutations in the target sequence, the primermay not be connected with the target sequence precisely. The nucleotidespecial structure near the mutation has been varied so as that LCR maynot be carried out and the amplification products may also not begenerated. At present, the method is mainly used in the research anddetection on the point mutation, such as the diagnoses of polymorphismsand products of single base hereditary diseases, research on specificidentification of microorganisms and point mutations in the cancergenes.

RCA is divided into two types: linear amplification and exponentialamplification. The former may only be applicable for annular nucleicacid amplification, whose products are a large number of DNA singlestrands of the repeated sequences complementary to the annular DNA. Thistechnique may be suitable for specific signal detection on micro-arraysor in the solid-phase forms. In exponential amplification, theamplification products may also act as the templates thereby increasingamplification products exponentially. This technique may also be usedfor the non-annular DNA amplification. The specificity of RCA is veryhigh, thus it can be used for mutation detection and SNP identification.Its use can be integrated with the fluorescent real-time detection,thereby enabling broad use of the technique.

TMA is amplification of RNA or DNA utilizing reverse transcriptase andT7 RNA polymerase under the isothermal conditions. In TMA reversetranscription of the target is accomplished by the action of the reversetranscriptase under the guidance a primer. The H activity of RNA reversetranscriptase degrades the RNA in the DNA-RNA hybrid chain therebypermitting the synthesis of double stranded DNA which can further betranscribed into thousands of RNA sequences under the action of T7 RNApolymerase. These RNAs may also act as the templates for next cycle. Thewhole TMA reaction is one autocatalytic process. The specificity of thismethod is high as is sensitivity. The reaction conditions are simple andthe amplification efficiency is high. TMA does not require specialamplification instruments and the whole reaction may be carried out in 1test tube thereby reducing environmental pollution.

Amplification relying on the nucleic acid sequences, calledself-sustained sequence replication (3SR), is used primarily for RNAdetection. The reaction depends on the reverse transcriptase, T7 RNApolymerase, nuclease H as well as two special primers. The 3′ end of theprimer I, is complementary with the target sequence and its 5′ endcontains T7 RNA polymerase promoter for cDNA synthesis. The sequences ofthe primer II are complementary with the 5′ end of the cDNA. During thereaction primer I is annealed to the RNA template to catalyticallysynthesize cDNA under the action of the reverse transcriptase. The RNAis then hydrolyzed by the nuclease H to form single-stranded DNA. PrimerII is annealed to the 5′ end of the cDNA and a second DNA strand issynthesized thereby forming a double-stranded DNA containing the T7 RNApolymerase promoter. Reverse transcriptase is used to transcribe a newRNA strand that is identical to the sample RNA sequence. Each new RNAstrand may also act as the template to synthesize cDNA. The process maybe repeated to form more RNAs and cDNAs. The operation is simple; nospecial instrument is required; no temperature cycling is required.Amplification may not be effective if the double stranded DNA has no anypromoter sequence so if the reaction specificity is increased greatly.This technology is suitable for detecting and quantitatively analyzingspecific RNA and also applicable for amplifying double stranded DNA.Therefore, it may be applied widely in the clinic.

SDA relies on the use of restriction endonuclease and DNA polymerase.SDA requires single-strand DNA template preparation in which DNAfragment of interest is generated in which the two ends of the fragmentinclude enzyme sites for SDA cycling. A primer containing therestriction endonuclease identification sites is combined with thesingle-stranded target molecules to form double-stranded DNA withsemi-phosphorylation sulfation sites by the action of DNA polymerasewhich has no excision enzyme activity. The unprotected primer chain iscut by the restriction endonuclease; whereas, the modified targetfragments remain intact and the DNA polymerase starts the extension atthe notch location and replaces the downstream sequences so as togenerate another DNA single strand whose notch may be opened by therestriction endonuclease. Such opening notches, polymerization andreplacement procedures are recycled repeatedly thereby generating alarge number of complementary strands of target molecules. SDA has thehigh sensitivity and can rapidly amplify single-stranded molecules;however, its application range is restricted because of the complexityof the target sequence preparation and detection method limitations.

LAMP is mainly made up of the Bst large-fragment DNA Polymerase and twopairs of special internal primers (FIP being made up of F1C and F2; BIPbeing made up of BIC and B2) and one pair of the external primer (F3 andB3). The F2 sequence of the FIP primer is coupled with the complementarysequence in the target DNA and the loop strand displacement reaction maybe started. The F3 primer is complementary with the F3C area in thetemplate to bring about and synthesize the double strands of thetemplate DNA so as to crowd out the DNA single strand introduced by FIP.In the meantime, the BIP primer is combined with the crowded out singlestrand hybridization so as to open the formed annular structure. Thenthe B3 primer is coupled with the base at the BIP outer side to form thenew complementary strand under the action of the polymerase. There arethe complementary sequences in the both ends of the displacedsingle-stranded DNA so as that the self base coupling may occur to formthe dumbbell DNA structure, which may act as the starting structure forLAMP reaction to recycle and extend and a large number of DNA sequencesare generated repeatedly and alternatively to form the amplificationproducts, which are stem-loop structure DNA with many loops and in thecauliflower shape. LAMP is highly specific and highly sensitive.Detection of pathogenic microorganisms using LAMP can be bothqualitative and quantitative as there is a linear relation between thequantity of magnesium pyrophosphate precipitation generation and thequantity of DNA generated. LAMP has a simple experimental setup and theexperiments are isothermal, thus only an ordinary water bath or otherdevices which can act as the stable heat source may be required. Thismethod may have applicability for scientific research work as well as aroutine detection tool.

TAS is primarily used for the amplification RNA. It utilizes reversetranscriptase, T7 RNA polymerase and nuclease H as well as two specialprimers. The 3′ end of the primer I is complementary with RNA foramplification and its 5′ end contains the promoter information of T7 RNApolymerase. The reverse transcriptase synthesizes cDNA by using primer Ias the starting point. Primer II is complementary with the 3′ end ofthis cDNA and is used to synthesize the second strand of the cDNA. T7RNA polymerase transcribes RNA which is the same as the RNA foramplification by taking the double stranded DNA as the template, whichmay be the template for the next round reaction. The TAS is with highamplification efficiency and its specificity is high; whereas, itscycling processes are complicated and the reverse transcriptase andT7-RNA polymerase may be added repeatedly; therefore, its further studywill be carried out.

HDA is a method that simulates natural DNA duplication, as it usesunwindases to separate DNA strands. HDA may be carried out under thesame temperature so as to optimize synthesis thereby reducing cost andpower consumption that a thermal cycler would require. The presentinvention provides a method of isothermal amplification and nucleic aciddetection in which one kind of strand displacement DNA polymerase(preferably Bst DNA polymerase) can be maintained for dozens of minutesat some certain constant temperature (about 62° C.), to carry outnucleic acid amplification reactions. Therefore, the methods of thepresent invention provide rapid nucleic acid amplification may becarried out quickly and effectively and in which only one simplethermostatic apparatus is required to carry out all amplificationprocesses so as to greatly decrease the complexity of the reaction (Nothermocycler required). The methods of the present invention also couplenucleic acid detection testing strip detection with cross primingamplification so as to develop one new rapid nucleic acid detectionmethod which enable amplification and detection processes to beaccomplished easily and simply. Template thermal denaturation, long-timetemperature cycling, tedious electrophoresis and other processes are nolonger required. The methods of the present invention are specific,simple and quick and may be applied broadly, with applications includingdiagnoses on the molecules directly related to human genetic diseases,detection of pathogenic microorganisms, estimation on the tumor orcancer diagnoses and prognosis and microorganism typing. Some isothermalamplification methods require initial denaturation of target DNA(Genomic DNA) at higher temperature before the isothermal reaction. CPAdoes not require initial thermal denaturation as it is truly anisothermal method.

SUMMARY OF THE INVENTION

The present invention relates to novel technology and methods for theamplification of nucleic acid sequences. More particularly, the presentinvention relates to methods of amplification of nucleic acids byutilizing cross priming isothermal amplification. Further the presentinvention relates to methods of marking the amplification targetsequence during the amplification reaction and rapid detection of thetarget sequence. The present invention also relates to the use of themethods in reagent kits for the rapid detection of nucleic acidsequences of pathogenic microorganisms such as bacteria, and viruses.The amplification and detection methods of the present invention canalso be utilized in the detection and diagnosis related to human geneticdiseases.

Cross Priming Amplification (CPA) uses multiple cross-linked primers,typically from 3 to 8 primers which can be paired, in which DNA targetsequence is amplified at one constant temperature, using a simpleheating device such as a water bath or a dry incubator. The number ofprimer/detector can be variable, according to the purpose andoptimization. The cross sequence can be designed for primer/primercross, or primer/detector cross, or other formats to satisfy differentapplications.

The detection of amplified products may be performed on a lateral flowstrip which may be housed in a sealed plastic device such as thatdescribed in US Patent Publication No. 2009/0181388A1, which preventsthe leakage of amplicons. During the reaction, the amplificationproducts are hybridized and labeled simultaneously, thereby making thelabeled amplified target ready for detection in a crosscontamination-proof lateral flow DNA strip device which provides avisual display read-out of the assay results.

In general CPA involves the generation of cross priming sites, crosspriming amplification and generation of detectable products. Forwardcross primer sense (PFs) and reverse cross primer anti-sense (PRa)primers are designed with 5′ sequences identical to each other's primingsequence. Displacement primers are designed that are located upstream ofthe cross primers. In preferred embodiments the concentration ofdisplacement primers are lower than that of the cross primers. DNApolymerase (in preferred embodiments Bst) extends the cross primer, andextend the displace primer. The extension of displacement primerdisplaces the cross primer extension strand, with a defined 5′ end. Asimilar extension/displacement mechanism on this new strand adds anotherpriming site, PFa on the other strand and also creates the other definedend.

The displaced strand contains newly introduced priming sites on bothends, and serves as template with priming sites for both cross primerson its 3′ end. A new priming site is introduced after each round ofextension/displacement, resulting in multiple primer binding sites whichaccelerate the amplification process.

The intermediate and end products are mixed in that they have differentlengths, and may have many forms of secondary structures (singlestranded, double stranded or partial double stranded). Detector probesor primers are used to probe for the target sequence. In a preferredembodiment one primer is extendable while the other is not extendableand these primers hybridize to the amplification products. Products thatare linked to both detectors are detectable, and in a preferredembodiment the detection mechanism utilizes a lateral flow stripdetection platform.

CPA utilizes the strand displacement function of DNA polymerase (such asBst, Klenow, Vent exo-DNA polymerase) to denature double stranded DNA.The use of such polymerase is shared by most isothermal amplificationmethods, including SDA, LAMP, CPA, RCA (Rolling Circle Amplification),HDA (Helicase Dependent Amplification).

SDA, LAMP and CPA all utilize a 5′ tail for some primers, but whereasthe purpose of 5′ tail in SDA is to introduce a nicking enzymerecognition site to the target and the purpose of 5′ tail in LAMP is tointroduce a sequence to form a loop in the target, the primary purposeof 5′ tail in CPA is to introduce additional priming sites at both endsof the target.

In SDA, the DNA extension mainly relies on nicking to create a free 3′end, annealed to the template strand for the synthesis of new DNA. InLAMP, the DNA extension mainly relies on the forming of self foldingloop, to anneal the free 3′ end of the loop to the template strand forthe synthesis of new DNA. In CPA, the DNA extension mainly relies on theannealing of multiple primers to multiple priming sites of both strandsto drive the synthesis of new DNA.

The methods of the present invention includes methods for amplifying atarget nucleic acid sequence comprising:

a) designing at least a first cross amplification primer and a secondcross amplification primer wherein the cross amplification primerscomprise a hybridization sequence and an interchanging sequence and atleast a first displacement primer and a second displacement primerwherein the first displacement primer is located 5′ to the first crossamplification primer and wherein first displacement primer is located 5′to the first cross amplification primer;

b) generating cross priming sites by introducing the cross amplificationprimers and the displacement primers to a target sequence in thepresence of a DNA polymerase under isothermal conditions such that crosspriming hybridization sites are introduced into the ends of the targetnucleic acid sequence thereby producing a target nucleic acid sequencecontaining cross amplification primer sites; and

c) amplification of the target nucleic acid sequence containing crossamplification primer sites through repeated hybridizations andextensions of the cross hybridization primers.

The methods of the present invention include methods for detecting thepresence of a target nucleic acid sequence comprising:

a) designing at least a first cross amplification primer and a secondcross amplification primer wherein the cross amplification primerscomprise a hybridization sequence and an interchanging sequence and atleast a first displacement primer and a second displacement primerwherein the first displacement primer is located 5′ to the first crossamplification primer and wherein first displacement primer is located 5′to the first cross amplification primer;

b) generating cross priming sites by introducing the cross amplificationprimers and the displacement primers to a target sequence in thepresence of a DNA polymerase under isothermal conditions such that crosspriming hybridization sites are introduced into the ends of the targetnucleic acid sequence thereby producing a target nucleic acid sequencecontaining cross amplification primer sites; and

c) amplification of the target nucleic acid sequence containing crossamplification primer sites through repeated hybridizations andextensions of the cross hybridization primers;

d) introducing a first detection primer labeled with a first marker anda second detection primer labeled with a second marker to the amplifiedtarget nucleic acid sequence containing cross amplification primer siteswherein the introduction of the first and second detection primersproduces double stranded nucleic acid molecules containing both markers;and

e) detecting the double stranded nucleic acid molecules containing bothmarkers.

The present invention also provides for kits for detecting pathogenicmicroorganisms, environmental microorganisms, microorganism typing,infectious disease pathogens for detecting human, animals or plants,infectious disease pathogens for detecting foods or biological weapons,the detection human genetic diseases or health risk genes comprisingcross amplification primers, displacement primers, detection primers andDNA polymerase as well as nucleic acid strip detection which may beplaced within a contamination-free device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—A schematic diagram of the basic concept of cross primeramplification.

FIG. 2—A schematic diagram of the basic primer design of a preferredembodiment of the cross primer amplification method of the presentinvention, including the cross amplification primer and detection primersite and sequence arrangement.

FIG. 3A—A schematic diagram of a preferred embodiment of the initiationphase of the cross priming amplification method including the generationof cross priming sites and defined ends.

FIG. 3B—A schematic diagram of a preferred embodiment of theamplification phase of the cross priming amplification method using alinear structure form which generates multiple priming sites.

FIG. 3C—A schematic diagram of a preferred embodiment of theamplification phase of the cross priming amplification method using asecondary structure forms, multiple primer binding and self fold andextension multiple priming sites.

FIG. 4—A schematic diagram of the generation amplified products and thedetection of amplified products which are double labeled.

FIG. 5—A schematic diagram outlining one method of detecting theamplification products by using the nucleic acid detection testingstrips

FIG. 6—The results of detecting the amplification products of Chlamydiatrachomatis by testing strips.

FIG. 7—The results of detecting the amplification products ofMycobacterium tuberculosis by testing strips.

FIG. 8A—A schematic diagram of single crossing amplification

FIG. 8B—A target sequence, primer location and primer design forMycobacterium tuberculosis in which an XbaI site is inserted into crossprimer between 1s and 2a.

FIG. 8C—Amplification products with different primer combinations. Lane1: 1s, 2a and 3a; Lane 2: 1s, 2a, 3a, 4s and 5a; Lane 3: 1s, 3a, 4s and5a; Lane 4: 1s, 2a, 4s and 5a; Lane 5: 2a, 3a, 4s and 5a; (-) indicatesno target control for the corresponding reaction. The system works withminimum of 3 primers, and at least 1 of these to be cross primer. The 2arrows point to the 2 smallest amplification products (1s/2a product and1s/3a product, respectively), which are basic units as shown in FIG. 2 aand the sequencing data (FIG. 2 e). Note that in lane 3, the 1s/2aproduct is missing, and in lane 4, the 1s/3a product is missing. Lane 5showed no product when the cross primer 1s is not present.

FIG. 8D—The CPA amplification product digested by restriction enzyme XbaI. Lane 1, CPA amplification product not digested; Lane 2, CPAamplification product digested by Xba I. High molecular weight productswere reduced, indicating repetitive fragments, The digestion is notcomplete possibly due to the heterozygous secondary structures of theamplification products amplification products.

FIG. 8E—Sequencing of CPA amplification products. The 2 bands indicatedby arrows at FIG. 8C were excited, cloned and sequenced. The sequencescorrespond to the final products illustrated in FIG. 8A. Theamplification products with higher molecular weight are tandem repeatsof these basic units (data not shown).

FIG. 9A-A schematic diagram of a mechanism of Cross PrimingAmplification (double crossing) High concentration Cross Primer 1sanneal and extend, lower concentration Displace Primer 3s anneal andextend later, displacing the down-stream strand. The displaced strand is5′ defined, with a new primer binding site 3) added at the 5′ end; CrossPrimer 2a anneal and extend, Displace Primer 4a displace the strand. Thedisplaced strand is now 3′ and 5′ defined, with another primer bindingsite added at the 5′ end, the single stranded DNA may form secondarystructures; 4) Both Cross Primer 1s and Cross Primer 2a can anneal tothe 3′ of the template. The extension product by Cross Primer 2a iselongated by adding another priming site at the 5′ end; 5) Similar tostep 4 with the other strand; 6), 7) and 8) With each round of extensionand displacement, the amplicon is elongated, with repetitive addition ofpriming sites. The repeated priming sites allow multiple primerannealing and extensions, facilitating the amplification. The repeatedsequences also form secondary structures and “branched” DNA, helping thetemplates stay at single stranded structures. The amplicons at thisstage are highly heterozygous, differing in lengths and structures.Multiple DNA synthesis may occur simultaneously on the same template.

FIG. 9B—Gel image of heterozygous amplification products

FIG. 9C—HPV target sequence and CPA primer design, displacement primersnot shown. The primer tags were designed for identification of eachprimer location in the sequenced amplicon. The amplification productswere cloned and 30 colonies were selected and sequenced. One of CPAamplicon sequences was shown. Note that the cross primers 1s and 2a areoverlapping each other.

FIG. 10A: Schematic of mechanism of double primer crossingamplification.

FIG. 10B: Schematic of Cross Priming Amplification (double crossing)

FIG. 10C: Gel image of heterozygous amplification products

FIG. 10D: Sequencing of an amplification product

FIG. 11A: Schematic of Single Crossing Amplification

FIG. 11B: Target sequence, primer locations and primer design

FIG. 11C: Gel of Amplification products with different primercombinations

FIG. 11D: Gel of restriction digestion of amplification products

FIG. 11E: Sequence analysis of amplification products Legend for FIG.11:

FIG. 11A: Mechanism of single crossing amplification.

FIG. 11B: Target sequence, primer locations and primer design. Note thatan Xba I site (TCTAGA) was inserted into the cross primer between 1s and2a.

FIG. 11C Amplification products with different primer combinations. Lane1: 1s, 2a and 3a; Lane 2: 1s, 2a, 3a, 4s and 5a; Lane 3: 1s, 3a, 4s and5a; Lane 4: 1s, 2a, 4s and 5a; Lane 5: 2a, 3a, 4s and 5a; (-) indicatesno target control for the corresponding reaction. The system works withminimum of 3 primers, and at least 1 of these to be cross primer. The 2arrows point to the 2 smallest amplification products (1s/2a product and1s/3a product, respectively), which are basic units as shown in FIG. 2 aand the sequencing data (FIG. 2 e). Note that in lane 3, the 1s/2aproduct is missing, and in lane 4, the 1s/3a product is missing. Lane 5showed no product when the cross primer 1s is not present.

FIG. 11D The CPA amplification product digested by restriction enzymeXba I. Lane 1, CPA amplification product not digested; Lane 2, CPAamplification product digested by Xba I. High molecular weight productswere reduced, indicating repetitive fragments, The digestion is notcomplete possibly due to the heterozygous secondary structures of theamplification products amplification products.

FIG. 11E: Sequencing of CPA amplification products. The 2 bandsindicated by arrows at FIG. 2 c were excited, cloned and sequenced. Thesequences correspond to the final products illustrated in FIG. 2 a. Theamplification products with higher molecular weight are tandem repeatsof these basic units (data not shown).

FIG. 12A: Gel of different reaction temperatures.

FIG. 12B: Gel of Sensitivity test for TB-CPA.

FIG. 12C: Gel of Specificity of TB-CPA.

FIG. 12D: Gel of time course of single crossing amplification.

FIG. 12E: Gel of negative test for TB-CPA

FIG. 13: Schematic of Assay Design Flexibility

FIG. 14: Theoretical Schematic of assembling of separated sequences

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel technology and methods for theamplification of nucleic acid sequences. More particularly, the presentinvention relates to methods of amplification of nucleic acids byutilizing cross priming isothermal amplification. Methods of the presentinvention named cross priming amplification utilize at least 6 types (3pairs) of specific primers which are designed according to 6 areas ofthe target nucleic acid. The amplification methods may be carried outunder constant temperature (isothermal) by using chain replacement DNApolymerases such as, but not limited to Bst DNA polymerase. Theprocesses of the present invention therefore may not require templatethermal denaturation or temperature cycling.

A preferred embodiment of the cross priming amplification methods of thepresent invention include the following procedures:

Primer design—Primer design may include one pair of cross amplificationprimers, one pair of displacement primers and one pair of detectionprimers.

Starting phase—Cross priming hybridization sites may be introduced intoeach end of the amplification target sequence. A fixed end may also begenerated so as to prepare the template for rapid amplification.

Amplification phase—The amplification may be carried out in either thelinear structure or secondary structure mode. Several primerhybridization sites may be generated and are carried out through therepeated hybridizations and extensions of the primers and theself-hybridization folding and extensions of the amplification productsso as that several amplification reactions may be carried outsimultaneously in the same template.

Detection of the products generated—Detection primers may be used tosynthesize dually-marked DNA double stranded molecules for detection bytaking the amplification products containing a large number of detectionsequences which are generated during the amplification phase as thetemplate. The amplification products may then be detected by using thenucleic acid detection testing strips.

Primer design may include one pair of cross amplification primers, onepair of displacement primers and one pair of detection primers. Inpreferred embodiments of the methods of the present invention one pairof cross amplification primers (cross amplification primer F and crossamplification primer R) are synthesized. Cross amplification primer Fand cross amplification primer R may be comprised of three segments:

1) Hybridization sequences—these are base sequences which hybridize withthe template with high specificity for the amplification extension.

2) Connectors—nucleotides used to connect two different sequences in theprimer, generally 1-3 mononucleotides.

3) Interchanging sequence—the hybridization sequence of the crossamplification primer F, which may also be the 5′ end sequence of crossamplification primer R; in like fashion, the hybridization sequence ofthe cross amplification primer R may act as the 5′ end sequence of crossamplification primer F. (FIG. 2).

One pair of displacement primers (displacement primer F and thedisplacement primer R) may be synthesized in which displacement primer Fis the forward outer primer that is complementary with the antisensestrand of the target gene and displacement primer R is the 5′ outerprimer that is complementary with the sense strand of the target gene.The displacement primer F and the displacement primer R are primarilyused for lengthening the strands of the displacement cross amplificationprimer during the starting phase in the isothermal amplificationreaction. (FIG. 2).

One pair of detection primers (the detection primer F and the detectionprimer R) may be synthesized in which the detection primer F is thebackward inner primer that is complementary with the sense strand of thetarget gene and the detection primer R is the forward inner primer thatis complementary with the antisense strand of the target gene. The pairof primers are marked by at least one different marker (preferably ahapten antigen), respectively, thus when amplifying the target sequencethe products of that amplification can be detected by the presence ofthe dual antigen (FIGS. 2, 4, 5).

During the starting phase cross priming hybridization sites may beintroduced into the each end of the amplification target sequence andfixed ends may be generated so as to prepare desired template for rapidamplification. CPA utilizes the strand displacement function of DNApolymerase (such as Bst, Klenow, Vent exo-DNA polymerase) to denaturedouble stranded DNA. To permit hybridization between the forwardamplification primer F and the nucleic acid target molecules the primersare designed such that there are hybridization identification sequencesof the backward primer R in the 5′ end of the forward primer F. Thedouble stranded nucleic acid may be synthesized by the DNA polymeraseextension forward amplification primer F with displacement function. Tocause the hybridization between the forward displacement primer F andthe nucleic acid target molecules. To use the DNA polymerase extensiondisplacement primer F with the displacement function and the extensionstrand of the displacement forward amplification primer F to generatethe fixed forward strand 5′ end.

The hybridization may be carried out between the displacementamplification primer extension strand as the template and the backwardamplification primer R, at whose 5′ end there is the hybridizationidentification sequence of the forward primer F. The double strandednucleic acid may be synthesized by the DNA polymerase extension backwardamplification primer R with displacement function. The hybridization iscarried out by using the backward displacement primer R and nucleic acidtarget molecules.

To use the DNA polymerase extension displacement primer R with thedisplacement function and the extension strand of the backwardamplification primer R to generate the fixed backward strand 5′ ends.Both ends of the generated backward amplification primer R extensionstrand have been fixed at this time; the hybridization sequences of theamplification primers F and R are introduced into the 3′ end and the 5′end, respectively, so as to act as the templates for rapidamplification. (FIG. 3A)

Amplification may be carried out in either a linear structure (FIG. 3B)and/or secondary structure mode (FIG. 3C). Multiple primer hybridizationsites are generated and are amplified throughout the repeatedhybridizations and extensions of the primers and the self-hybridizationfolding and extensions of the amplification products so as that severalamplification reactions may be carried out simultaneously in the sametemplate. Primer extensions at the ends displace the extension strands,which may be again be used as the template to participate in the nextround of amplification so as to greatly enhance the amplification speed.

In the linear structure mode, one new primer hybridization sequence maybe introduced into the amplification primer for every extension and theamplification products may be lengthened along with every round ofamplification. Primer hybridization opportunities are increased becauseof the increased synthesizing speed of short strand products such thatthe most of the final products may be the short strand products. Inother words, the products by using the cross priming amplificationmethod may be the mixture of amplification products of differentlengths, in which the long stranded amplification products may providethe multiple primer hybridization sites for further amplificationthereby enhancing the amplification speed and the short strandedamplification products may be the main objects for the final detection.(FIG. 3A).

In the secondary structure mode, the amplification products may formvarious complicated secondary structures through self-folding due to thepresence of repeated complementary sequences in the long strandamplification products. These amplification products can be longer andthe structures more complicated through self extension and becausesingle strands may provide primer hybridization for synthesis of newamplification products. The linear structure and the secondary structuremay be interchanged. The single-stranded linear structure and secondarystructure molecules may be interconnected through hybridization so as toform huge DNA hybridization complexes. Therefore, the products in thecross priming amplification method are very complicated as determined bygel electrophoresis. Different length DNAs may be seen and huge DNAhybridization complexes are located in the sampling wells of gelelectrophoresis as they have almost no mobility.

After amplification the products can be detected in various fashions. Ina preferred embodiment detection primers may synthesized each containingone or more different markers. The resulting dually marked DNA doublestranded molecules can be detected by taking the amplification productscontaining a large number of detection sequences which are generatedduring the amplification phase. Most of such dually marked DNA doublestranded molecules are relatively short amplification final products;whereas, the relatively large amplification products or hugehybridization complexes contains the hybridization sites of thedetection primers, which may obtain the dually marking through thedetection primer hybridization; thus they may be detected. (FIG. 4).

The amplification products of the dually marked target sequences may bedetected through agarose gel electrophoresis or nucleic acid detectiontesting strips. One embodiment of the detection principal of the nucleicacid testing strip is shown in FIG. 5.

The schematic for a single crossing assay for CPA is shown in FIG. 11A.In the single crossing assay, the primer set is designed to increase theabundance of small amplicons while still providing the featuresnecessary for overall amplification of the target sequence in thetemplate. The primary function of cross primer 1s is identical to thatin the double crossing assay: to allow strand displacement and toincorporate a second defined priming site in the 5′ end of the resultingproduct. Primers for the opposite strand each consist of a singlesequence complementary to the template, and are chosen so that they bindin tandem on the template, in essence providing a region of nickeddouble stranded DNA that can be extended by a strand displacing DNApolymerase. The displaced single stranded product from both strands arecomplementary at their 5′ and 3′ ends by virtue of the introduction ofthe 2a sequence at the 5′ end of the top strand and the 2s sequence atthe 3′ end. These single stranded DNAs are able to form hairpin-likestructures stabilized by the 19 base pair double helix formed between 2aand 2c, and can act as templates for further extension reactions (FIG.11A).

FIG. 11B shows a set of five primers to the IS6110 region ofMycobacterium tuberculosis genomic DNA for a single crossingamplification reaction. Amplification reactions were carrier out in thepresence of template DNA using various combinations of the 5 primers(FIG. 11C). The two arrows point to the 1s/2a amplification product(top) and the 1s/3s amplification product (bottom), which are the basicunits shown in FIG. 10A. Both products are obtained using solely the 1s,2a and 3a primers (lane 1), but the yield is enhanced when primers 4sand 5a are also included in the reaction (lane 2). When primer 2a isomitted from the mix (lane 4). If the cross primer 1s is not present inthe reaction, there are no products produced from the mixture of fourprimers (lane 5). These result demonstrate that the system works with aminimum of three primers as long as one of these primers is a crossprimer. (e.g. primer 1s).

The reaction scheme does not predict the formation of the highermolecular weight amplicons observed in a single crossing reactionproducts shown in FIG. 11C. To determine whether these productsrepresent tandem copies of the basic amplicons, the reaction productswere digested with Xba I, which is introduced in the design of the 1sprimer. The higher molecular weight products of the single crossingamplification reaction are reduced in size upon digestion with Xba I(FIG. 11D) as are the 1s/3a and 1s/2a basic products. The incompletedigestion of the higher molecular weight fragments may be the result oftheir ability to form a variety of secondary structures which mightinterfere with restriction enzyme cleavage at some Xba I sites.

The two bands corresponding to the smallest amplification products (FIG.11C) were excised from the gel, ligated into a TA vector and sequenced.The sequences obtained correspond to the predicted 1s/3a and 1s/2a finalproducts (FIG. 11E). The amplification products with higher molecularweight were cloned and sequenced and are tandem repeats of the 1s/3a and1s/2a basic units.

CPA offers a great deal of flexibility through different primer designstrategies and by controlling the ratio of the various primers (FIG.13). Of particular interest are the strategies for asymmetricamplification, multiplex amplification coupled to specific detection anddetection of separated target areas by encouraging template switchingand DNA shuffling in a controlled fashion. This last application ofinterest in designing amplification protocols to detect pathogens thatundergo rapid mutation and have only a small conserved sequence areawidely dispersed in their genomes. CPA combined with careful primerdesign can result in the production of initial extension products thatcombine the dispersed conserved regions in a tandem fashion thusproducing a template for further amplification and detection (FIG. 14).

CPA can be used for single universal primer detection of multipletargets. CPA may be designed to detect different pathogenssimultaneously with a universal tail. Two sets of specific primers witha universal tail are used to amplify the specific pathogen with lowconcentration in the initial stage. The amplicons with the universalsequence at both 5′ and 3′ ends are further amplified by a singleuniversal primer, which is at a higher concentration. Specific ampliconsfrom different targets are (if present in the sample) may “fuse” to fora heterogenous template and be amplified and distinguished by a specificdetector probe.

The optimal temperature for an isothermal amplification assay mustbalance binding of the primer to the template strand with elongationactivity of DNA polymerase. Single crossing amplification reactions werecarried out at different temperatures using M. tuberculosis DNA as withthe appropriate primers. Roughly equivalent levels of amplicons wereobserved to form in a template-dependent manner at temperatures rangingfrom 63° C. to 68° C., but failed to form 70° C. (FIG. 12A).Temperatures below 63° C. gave little to no product. A reactiontemperature 63° C. was selected as the standard temperature for the restof the CPA assays conducted.

The minimal level of template required for detection by the CPA assaywas determined by conducting single crossing amplification assays withserial dilutions of M. tuberculosis bacteria cells. FIG. 12B showsamplicons were observed when as few 4 bacterial cells were present inthe amplification reaction, indicating that the CPA assay is able toproduce specific amplicons from just a few cells.

In the polymerase chain reaction, the reduction in the background noiseof non-specific products derived from mis-priming events, orprimer-primer interactions is controlled by restricting the number ofthermal cycles. In an isothermal amplification reaction, totalincubation time will influence the production of specific vs.nonspecific amplicons. Single crossing amplification reactions wereincubated from 60 minutes to 210 minutes in the absence of template(FIG. 12C). Non-specific amplicons appeared after 180 minutes with abanding pattern distinct (lane 5) from the pattern obtained after a 60minute amplification in the presence of the M. tuberculosis DNA template(lane 7). FIG. 12D shows a time course for the production of specificamplicons in a single crossing amplification assay conducted with 10⁴copies of template DNA. From the intensity of the banding pattern it isapparent that the reaction reaches completion after 28 minutes, while a60 minute incubation in the absence of template DNA (lane N) does notyield any amplicons.

The specificity of the single crossing amplification reaction was testedusing bacterial cells from a variety of mycobacterial species (FIG. 12E). Amplification products were only observed in the reactions thatcontained either M. tuberculosis cells (lane 13) or a plasmid templatecontaining M. tuberculosis DNA target region (lane 14), demonstratingthat primers used in the assay and the reaction mechanism combine with ahigh degree of specificity.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Detection of Chlamydia trachomatis

Since 1990, Chlamydia trachomatis has become the most common pathogen inurinary tract infections supplanting Neisseria gonorrhoeae. Along withthe gradually increasing of the Chlamydia trachomatis infection diseasesand corresponding complicating diseases, it has become greater threat tothe human reproductive health than ever. In 1995, the World HealthOrganization estimated that there were about 90 million Chlamydiatrachomatis patients all over the world, which were only lower thatthose of HIV infection patients. Therefore, to realize the rapidChlamydia trachomatis diagnosis has an important significance. Theinvention patent may be used to rapidly detect the DNA of the Chlamydiatrachomatis, whose concrete design on the corresponding primers may belisted as follows: we select the DNA sequences of the Chlamydiatrachomatis and the gene fragments for amplification; moreover, thespecificity primer may be designed according to the sequences.

The basic components of the amplification reactions are listed below:

Displacement primer F  0.05 μmol Displacement primer R  0.05 μmolCross amplification primer F  0.4 μmol Cross amplification primer R 0.4 μmol Detection primer F  0.2 μmol Detection primer R  0.2 μmoldNTP:  0.4 mmol Thermopol buffer (10x)  2 μL MgSO₄  8 mmol Betaine 1 mol Bst DNA Ploymerase  8 units Reaction total volume: 20 μLDisplacement primer 1- (SEQ ID 1) 5′-TTTGCCTTAACCCCACCATT-3′Displacement primer 2- (SEQ ID 2) 5′-CCTCTGAAGTCTTAAGCTTG-3′Cross Amplification primer F- (SEQ ID 3)5′-ATTAGTCAGATTTGTTTCCAACTTCCGGAGTTACGAAGA-3′Cross Amplification primer R- (SEQ ID 4)5′-TCCGGAGCGAGTTACGAAGATATTAGTCAGATTTGTTTCCAAC-3′ Detector F- (SEQ ID 5)5′-TACAAGAGTACATCGGTCAA-3′ Detector R- (SEQ ID 6)5′-GGGAGAAAGAAATGGTAGC-3

Optimization of the reaction time, temperature, primer content andmagnesium concentration were investigated. In each case the reactantswere held constant except for the parameter being investigated. Thereaction time for the amplification reaction were investigating using 66minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78minutes and 80 minutes. The optimal reaction time appears to be about 80minutes.

The reaction temperature for the amplification reaction was investigatedusing 54° C., 56° C., 58° C., 60° C., 62° C., 64° C. and 66° C., thesignals are enhanced with increasing reaction temperature up to about60° C. After about 60° C. the signal decreases thereby indicating thatthe optimal temperature for the reaction is around 60° C.

The concentration of primers F and R for the amplification reaction wereinvestigated using 0.8 μmol, 0.7 μmol, 0.6 μmol, 0.5 μmol, 0.4 μmol, 0.3μmol, 0.2 μmol and 0.1 μmol. The optimal concentration of the primersappears to be about 0.4 μmol.

The Mg²⁺ concentration for the amplification reaction was investigatedusing 10 mmol, 9 mmol, 8 mmol, 7 mmol, 6 mmol and 5 mmol. The optimalMg²⁺ concentration appears to be about 8 mmol.

The results of the detection of the amplification products by using thetesting strips is shown in FIG. 6.

Example 2 Detection of Mycobacterium tuberculosis

Mycobacterium tuberculosis (TB) viral DNA was used as template. The CPAreaction mixture contained five primers F3, B3, F1, F2, BIP,respectively. BIP consisted of B1c sequence complementary to the B1 andthe F1 sequence. F1 had labeled Biotin at its 5 end, and F2 labeled waslabeled with FitC at its 5′ end. Amplification conditions were optimizedfor temperature, primer and probe concentrations, enzyme units, Mg++concentration, buffer concentration, and reaction time. The optimizedreaction was carried out in a total of 20 μl contained 0.5 μM each BIP,F1 and F2, 0.05 μM F3 and B3, 0.8 mM each dNTP, 1M betaine(sigma), 20 mMTris-HCl (pH 8.8), 10 mMKCl, 10 mM (NH4)2SO4, 6 mM MgSO4, 0.1% TritonX-100, 8 U Bst DNA polymerase large fragment (New England Biolabs) andthe specified amounts of double-stranded target DNA. The mixture wasincubated at 66° C. for 1 h, without being heated at 95° C. for 5 min.After incubation, the amplified products were detected by nucleic aciddetection strip directly without opening the lid of the PCR tube.

(SEQ ID 7) AGGACCACGATCGCTCCGGCCACAGCCGTCCCGCCGATCTCGTCCAGCGCCGCTTCGGACCACCAGCACCTAACCGGCTGTGGGTAGCACCTCACCTATGTGTCGACCTGGGCAGGGTTCGCCTACGTGGCCTTTGTCACCGACGCCTACGCTCGCAGGATCCTGGGCTGGCGGGTCGCTTCCACGATGGCCA TBMPF2 (SEQ ID 8)5′-ACAGCCCGTCCCGCCAT-3′ TBMMRin-5B (SEQ ID 9)5′-TAGCAGACCTCACCTATGTGTC-3′ TBDF-5F2 (SEQ ID 10)5′-CTGGGCAGGGTTCGCCT-3′ TBBIP (SEQ ID 11)5′-TAGCAGACCTCACCTATGTGTC-T-TCGGTGACAAAGGCCACGT TBB3 (SEQ ID 12)5′- TCGGTGACAAAGGCCACGT-3′ TBF3 (SEQ ID 13) 5′-AGGACCACGATCGCTGATC-3′

The invention claimed is:
 1. A kit for detecting Mycobacteriumtuberculosis comprising: a) a primer set comprising SEQ ID 11 and two ormore primers selected from the group of primers consisting of SEQ ID 8,SEQ ID 9, SEQ ID 10 and SEQ ID 17; and b) a DNA polymerase.
 2. The kitof claim 1 further comprising a detection strip.
 3. The kit of claim 2wherein the detection strip is enclosed in a device that preventscontamination of the sample.
 4. The kit of claim 1 wherein the DNApolymerase is Bst DNA polymerase.